The goal of the project is to identify therapeutic candidates capable of eradicating recalcitrant chronic infections such as osteomyelitis, endocarditis, abscesses, and biofilms of indwelling devices. Many of these infections are essentially untreatable and lead to substantial morbidity and mortality. Biofilms harbor dormant persister cells that are highly tolerant to killing by currently available antibiotics which were developed against rapidly growing cells. Mechanisms of persister formation are poorly conserved and redundant. This suggests that the traditional drug discovery approach, developing inhibitors against a target, will not work. We reasoned that persisters can be killed by activating cellular proteolysis. Acyldepsipeptide (ADEP) activates the conserved ClpP protease, relieving it both from regulatory control and from the requirement to use ATP. Our preliminary studies with ADEP4 show that this compound has an exceptional killing activity against growing, stationary and biofilm cultures of S. aureus. ClpP is not essential, and a high-frequency null mutation leads to resistance. For this reason, development of ADEP4 was terminated. However, knowing the sterilizing potential of ADEP4, we decided to combine it with conventional antibiotics to solve the resistance problem. ADEP4 in combination with rifampicin eradicated MRSA in a mouse model of deep-seated infection, where conventional antibiotics had very little effect. However, ADEP4 is a large lipopeptide with poor oral bioavailability, limited solubility and rapid metabolism. The aim of this project is to develop more potent ClpP activators with improved pharmacological properties. We will use two approaches: produce superior ADEP analogs, taking advantage of the crystal structure of the S. aureus ClpP that we recently solved; and develop novel-based therapeutics through advanced structure and fragment based ligand design. We developed an efficient ADEP synthetic route and produced the first analogs with improved physiochemical properties. We also performed a virtual screen of fragments and obtained novel small molecules that have been extensively validated as activators of S. aureus ClpP. In this project, we will expand the structure-based design of ADEP analogs and will obtain effective small molecule-based ClpP activators. The new compounds will be analyzed for their ability to eradicate a number of important pathogens in in vitro and in vivo models of chronic infection. The mechanism of killing by ClpP activators will be examined to provide a better understanding of the unique killing by this type of molecules and to provide further information for therapeutic design. This multi-PI project is led by Dr. Richard Lee, a medicinal chemist, who will be responsible for producing new ClpP activators and Dr. Kim Lewis, a microbiologist, who will perform the antimicrobial evaluation against persisters.